Abstract
Purpose:
Diabetic retinopathy can cause abnormalities in the ganglion cell layer, which may contribute to visual impairment. Previously, we have shown that plasma kallikrein (PK) is present in vitreous samples obtained from patients with advanced diabetic retinopathy. In this study, we examine the effects of PK on the neuroretina in diabetic retinopathy in rodent models, and examine the direct effects of PK on neurons.
Methods:
Diabetes was induced in Sprague Dawley rats by IP injection with streptozotocin. The effects of intravitreal PK on ganglion cells were examined in rats with 4 weeks of diabetes subjected to intravitreal injection of activated PK (50ng). The effects of systemic PK inhibition on the neuroretina were examined in diabetic rats treated with the PK inhibitor (BPCCB) or vehicle alone continuously administered via subcutaneous Alzet osmotic pumps for 4 weeks. Retinal Brn3a (a ganglion cell marker) and caspase 3 levels were quantified by western blot and immunohistochemistry. The effects of PK on cultured cortical neurons were examined by measurements of lactate dehydrogenase release. The effects of PK on NMDA receptors (NR) were examined by western blotting and mutational analyses of NR.
Results:
We show that intravitreal injection of PK in diabetic rats significantly decreased Brn3a (PK injection group n=9, Vehicle injection group n=9, P<0.01) and increased caspase 3 (P<0.01) in retinal extracts harvested at 24 hrs post injection. Administration of the selective PK inhibitor (BPCCB) increased Brn3a (PK inhibitor group n=8, Vehicle injection group n=10, P<0.05) and decreased caspase 3 (P<0.01) level in diabetic retina compared to vehicle treatment. In vitro, we found that PK-induced cortical neuronal cell death requires the presence of NR. Using site directed mutagenesis, we found that the PK directly cleaves NR1 at Arg323 residue located in the extracellular N-terminal domain of the receptor and this cleave mediated the effect of PK on enhancing NMDA’s effects.
Conclusions:
These results demonstrate that intravitreal injection of activated PK in diabetic rats triggers retinal neuro-degeneration while inhibition of PK reduces ganglion cell loss in diabetes. Our findings suggest that PK-induced neuron injury is mediated by cleavage-dependent activation of NMDA receptor.
Keywords: 499 diabetic retinopathy •
695 retinal degenerations: cell biology •
615 neuroprotection